MFLP-94 The Eight Hour Reveal Method for Detecting Escherichia coli O157_H7 In Raw Beef and Environmental Samples
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1B27BAEB4E074E4CBAD0827000172FB8 |
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0.03 |
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页数： |
6 |
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日期： |
2012-3-2 |
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Published on the Food Directorate’s (Health Canada) website at http://www.hc-sc.gc.ca/food-aliment.,Government of Canada Gouvernement du Canada,Laboratory Procedure MFLP-94,September 2000,THE EIGHT HOUR REVEAL METHOD FOR DETECTING ESCHERICHIA COLI O157:H7,IN RAW BEEF AND ENVIRONMENTAL SAMPLES.,1. APPLICATION,The method is applicable to the detection of Escherichia coli O157:H7 from raw beef cubes, raw ground,beef, and environmental swabs to determine compliance with the requirements of Sections 4 and 7 of,the Food and Drugs Act.,2. DESCRIPTION,The method has been shown to produce satisfactory results with artificially contaminated raw cubed,beef and raw ground beef as well as environmental swabs from stainless steel in an AOAC study (8.1).,The Microbiological Methods Committee has approved this method for the detection of E. coli O157:H7,in raw beef and environmental swabs only. Other food products should be analysed using other,methods available in this Compendium.,3. PRINCIPLE,Following selective enrichment in the Reveal 8 hour broth, a portion of the sample enrichment is placed into,the sample port of the Reveal Device initiating flow. The Reveal Device contains antibodies with high,specificity to E. coli O157:H7 antigens. These antibodies are bound to colloidal gold and, separately, to a,solid support matrix. Any E. coli O157:H7 antigen present will bind to the gold conjugated antibodies forming,an antigen – antibody – chromogen complex. This complex flows across a lateral flow membrane and is,subsequently bound by antibody immobilized on the membrane. This causes the gold conjugate to,precipitate, forming a visible line and indicating a positive reaction. Proper test completion and flow is,indicated by a control line which forms further up in the test window and verifies a valid test run. Absence,of a control line invalidates the test. Record results at 15 minutes incubation time.,4. DEFINITION OF TERMS,See Appendix A of Volume 3, items 1,2,3,4.,5. COLLECTION OF SAMPLES,See Appendix B of Volume 3.,6. MATERIALS AND SPECIAL EQUIPMENT,1) Reveal Device (Neogen Corporation, phone: 1-800-234-5333, Fax: (517) 372-0108),2) Reveal 8 hour broth (Neogen Corporation),3) Biosynth BCM O157:H7+ chromogenic agar (Biosynth, Naperville, IL),4) Hemorrhagic coli agar,5) MacConkey sorbitol agar (Acumedia, Baltimore, MD),6) mHC agar or TCCSMAC agar (see MFLP-80),MFLP-94,- 2 - September 2000,7) Butterfield’s phosphate buffer (Fisher Scientific), or peptone water 0.1%,8) Incubators capable of maintaining 36EC.,9) Incubators capable of maintaining 42EC.,NOTE 1 : It is the responsibility of each laboratory to ensure that the temperature of the incubators or water,baths are maintained at the recommended temperatures. Where 35EC is recommended in text of,the method the incubator may be at 35 +/-1.0E C. Similarly, lower temperatures of 30 or 25 may be,+/- 1.0EC. However, where higher temperatures are recommended, such as 43 or 45.5EC, it is,imperative that the incubators or water baths be maintained within 0.5EC due to potential lethality,of higher temperatures on the microorganism being isolated.,10) ?Stomacher ?Colworth or equivalent,11) ?Stomacher ?bags,12) Micropipette (200 μl) and sterile tips,13) Control cultures,E. coli O157:H7 ATCC 35150; ATCC 43895,7. PROCEDURE,Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test,in accordance with the following instructions:,7.1 Handling of Sample Units,7.1.1 In the laboratory prior to analysis, keep sample units refrigerated (0-5EC) or frozen,depending on the nature of the product. Thaw frozen samples in a refrigerator, or under,time and temperature conditions which prevent microbial growth or death.,7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory,7.2 Preparation for Analysis,7.2.1 Have ready the Reveal 8 hour broth kept at 42 °C, see 9.1.,7.2.2 Clean the surface of the working area with a suitable disinfectant,7.3 Preparation of Sample,To ensure a truly representative analytical unit agitate liquids or free flowing materials until the,contents are homogeneous. If a sample unit is a solid, obtain the analytical unit by taking a portion,from several locations within the sample unit. To reduce the workload, the analytical units may be,combined for analysis. It is recommended that a composite contain not more than 500g.,7.4 Enrichment of Sample,7.4.1 Prepare aseptically a 1:10 dilution of the food, 25 g or mL (the analytical unit) into 225 mL,of the required enrichment broth, as indicated in Table 1. Place bag in the ?Stomacher?,and mix for 2 minutes at normal speed o……
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